CA1144920A - Cephalosporin antibiotics - Google Patents

Abstract

ABSTRACT
"Cephalosporin antibiotics"

Cephalosporin antibiotics of general formula (I) (wherein Ra and Rb, which may be the same or different, each represent a C1-2 alkyl group, or together with the carbon atom to which they are attached form a C3-6 cycloalkylidene group and Y ? represents a 1-carbamoyl-methylpyridinium group) exhibit broad spectrum antibiotic activity, the activity being unusually high against gram-negative organisms such as strains of Pseudomonas organisms.
Particularly effective compounds of formula (I) are (6R,7R)-7-[(Z)-2-(2-aminothiazol-4-yl)-2-(2-carboxy-prop-2-oxyimino)acetamido]-3-[(1-carbamoylmethylpyridinium-4-yl)thiomethyl]ceph-3-em-4-carboxylate and (6R,7R)-[(Z)-2-(2-aminothiazol-4-yl)-2-[(1-carboxycyclobut-1-oxyimino)acetamido]-3-[(1-carbamoylmethylpyridinium-4-yl)thiomethyl]-ceph-3-em-4-carboxylate. The invention also includes the non-toxic salts and non-toxic metabol-ically labile esters of compounds of formula (I), com-positions containing the antibiotic compounds of the invention and processes for the preparation of the antibiotics.

Description

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''Cephalosporin antibiotics"

This invention is concerned with cephalosporin compounds possessing valuable antibiotic properties.
The cephalosporin compounds in this specification are named with reference to "cepham" after J. Amer.
Chem. Soc., 1962, 84, 3400, the term "cephem" referring to the basic cepham structure with one double bond.
Cephalosporin antibiotics are widely used in the treatment of diseases caused by pathogenic bacteria in human beings and animals, and are especially useful in the treatment of diseases caused by bacteria which are resistant to other antibiotics such as penicillin compounds, and in the treatment of penicillin-sensitive patients. In many instances it is desirable to employ a cephalosporin antibiotic which exhibits activity against both gram-positive and gram-negative microorgan-isms, and a significant amount of research has been directed to the development of various types of broad spectrum cephalosporin antibiotics.
Thus, for example, in our British Patent Specifi-cation No. 1,399,086, we describe a novel class of cephalosporin antibiotics containing a 7~-(-etherified oxyimino)-acylamido group, the oxyimino group having the syn configuration. This class of antib~otic compounds is characterised by high antibacterial activity against a range of gram-positive and gram-negative organisms coupled with particularly high stability to ~-lactamases produced by various gram-negative organisms.
The discovery of this class of compounds has stimulated further research in the same area in attempts to find compounds which have improved properties, for example against particular classes of organisms, especially gram-negative organisms.
For example, in our British Patent Specification .,~

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No. 1,496,757, we describe cephalosporin antibiotics containing a 7~-acylamido group of formula R . C . CO . Nl 1-N RA
0. (CH2)m C (CH2)nCOOH (A) RB

(wherein R is a thienyl or furyl group; R and RB may vary widely and may, for example, be Cl 4 alkyl groups or together with the carbon atom to which they are attached form a C3 7 cycloalkylidene group, and m and n are each 0 or l such that the sum of m and n is 0 or l), the compounds being syn isomers or mixtures of syn and anti isomers containing at least 90~ of the syn isomer. The 3-position of the cephalosporin molecule may be unsubstituted or may contain one of a wide variety of possible substituents. These compounds have been found to have particularly good activity against gram-negative organisms.
Other compounds of similar structure have been developed from these compounds in further attempts to find antibiotics having improved broad spectrum antibiotic activity and/or high activity against gram-negative organisms. Such developments have involved variations not only in the 7~-acylamido group of formula (A) but also the introduction of particular groups in the 3-position of the cephalosporin molecule.
Thus, South African Patent Specification 78/1870 describes cephalosporin compounds wherein the 7~-acylamido side-chain is inter alia a 2-(2-aminothiazol-4-yl)-

2-(optionally substituted alkoxyimino)acetamido group~
In these compounds, the substituent in the 3-position may be selected from a wide variety of organic radicals including inter alia a group of formula -CH2SRX where Rx may be a heterocyclic group, e.g. a pyridyl group, . ..

which may be substituted by inter alia a carbamoylmethyl group. The specification contains, among numerous other examples, references to compounds in which the above-mentioned optionally substituted alkoxyimino group is a carboxyalkoxyimino or carboxycycloalkoxyimino group.
Belgian Patent Specification No. 836,813 describes cephalosporin compounds wherein the group R in formula (A) may be replaced by, for example, 2-aminothiazol-4-yl and the oxyimino group is a hydroxyimino or blocked hydroxyimino group. In such compounds, the 3-position of the cephalosporin molecule is substituted by a methyl group which may itself be optionally substituted by any of a large number of residues of nucleophilic com-pounds therein described. Examples of such residuesinclude the mercapto group which may be attached to a 5- or 6-membered heterocyclic ring containing from 1 to 4 heteroatoms selected from oxygen, sulphur and nitrogen. Amongst the examples given of such heterocyclic rings is the pyridyl group which may if desired be substituted, for example, by a lower alkyl group or a carbamoyl group. In this specification no antibiotic activity is ascribed to such compounds which are only mentioned as intermediates for the preparation of anti-biotics therein described.
Furthermore, Belgian Patent Specification No.852,427 describes cephalosporin compounds wherein the group R in formula (A) may be replaced by a variety of different organic groups including 2-aminothiazol-4-yl and wherein the oxygen atom in the oxyimino grouping is attached to an aliphatic hydrocarbon group which may itself be substituted by, for example, a carboxy group. In such compounds, the substituent at the 3-position may vary widely and may be inter alia an option-ally substituted heterocyclicthiomethyl group. Manyexamples of such groups are given in the specification including those in which the heterocyclic moiety of ~1~49~

the group is a 3- to 8- membered heterocyclic ring containing 1 to 4 nitrogen atoms, e.g. an imidazolyl, pyrazolyl, pyridyl, pyrimidyl or tetrazolyl group which may be substituted.
We have now discovered that by an appropriate selection of a small number of particula.r groups at the 7~-position in comhina-tion with an N-carbamoylmethylpyridinium-thiomethyl group at the 3-position, compounds havi.ng particularly good activity (described in more detail below) against a wide range of commonly encountered pathogenic organisms may be obtained.

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The present invention provides cephalosporin antibiotics of the general formula:

S ~ N ,H H S
/ C.CO.NH - ~ ~
N R o ~ N ~ CH2SY ~ (I) O.C.COOH
COO
Rb (wherein Ra and Rb, which may be the same or different, each represents a Cl 2 alkyl group or together with the carbon atom to which they are attached, form a C3 6 cycloalkylidene group and Y ~ represents an N-carbamoylmethylpyridinium group) and non-toxic salts and non-toxic metabolically labile esters thereof.
The compounds according to the invention are syn isomers. The syn isomeric form is defined by the configuration of the group Ra -O.C.COOH
Rb with respect to the carboxamido group. In this specifi-cation the syn configuration is denoted structurally as Z~

~'E~2 S N
.CO.NH
N Ra O~C.COOH
Rb It will be understood that the compounds according to the invention are geometric isomers and some admixture with the corresponding anti isomer may occur.
The invention also includes within its scope the solvates (especially the hydrates) of the compounds according to the invention. It also includes within its scope salts of esters of compounds of formula (I).
It will be appreciated that the N-carbamoylmethyl-pyridinium group may be attached to the sulphur atom via the 2-, 3- or 4-carbon atom of the pyridine ring.
The compounds according to the present invention may exist in tautomeric forms (for example in respect of the 2-aminothiazolyl group) and it will be understood that such tautomeric forms, e.g. the 2-iminothiazolinyl form, are included within the scope of the invention.
Moreover, the compounds of formula (I) depicted above may also exist in alternative zwitterionic forms, for example wherein the 4-carboxyl group is protonated and the terminal carboxyl group in the 7-side chain is deprotonated. Such zwitterionic forms and mixtures thereof are included within the scope of the present invention.
It will also be appreciated that when one of Ra and Rb in for~ula (I) represents a methyl group and the other represents an ethyl group, the carbon atom to which they are attached will comprise a centre of asymmetry. Such compounds are diastereoisomeric 4~'~0 and the present invention embraces individual diastero-isomers of these compounds as well as mixtures thereof.
The compounds according to the invention exhibit broad spectrum antibiotic activity. Against gram-negative organisms the activity is unusually high. This high activity extends to many ~-lactamase-producing gram-negative strains. The compounds also possess high stability to ~-lactamases produced by a range of gram-positive and gram-negative organisms.
Compounds according to the invention have been found to exhibit unusually high activity against strains of Pseudomonas organisms, e.g. strains of Pseudomonas aeruginosa as well as high activity against various members of the Enterobacteriaceae (e.g. strains of Escherichia coli, Klebsiella pneumoniae, Salmonella typhimuri _, Shigella sonnei, Enterobacter cloacae, Serratia marcescens, Providence species, Proteus mirabilis and especially indole-positive Proteus organisms such as Proteus vulgaris and Proteus morganii), and strains of Haemophilus influenzae.
The antibiotic properties of the compounds according to the invention compare very favourably with those of the aminoglycosides such as amikacin or gentamicin.
In particular, this applies to their activity against strains of various Pseudomonas organisms which are not susceptible to many existing commercially available antibiotic compounds. Unlike the aminoglycosides, cephalosporin antibiotics normally exhibit low toxicity in man. The use of aminoglycosides in human therapy tends to be limited or complicated by the relatively high toxicity of these antibiotics. The cephalosporin antibiotics of the present invention thus possess potent-ially great advantages over the aminoglycosides.
Non-toxic salt derivatives which may be formed from the compounds of general formula (I) include inor-ganic base salts such as alkali metal salts (e.g.
sodium and potassium salts) and alkaline earth metal salts (e.g. calcium salts); amino acid salts (e.g.

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lysine and arginine salts); organic base salts (e.g.
procaine, phenethylbenzylamine, dibenzylethylenediamine, ethanolamine, diethanolamine and N-methyl-glucosamine salts). Other non-toxic salt derivatives include acid addition salts, e.g. formed with hydrochloric, hydrobromic, sulphuric, nitric, phosphoric, formic and trifluoroacetic acids. The salts may also be in the form of resinates formed with, for example, a polystyrene resin or cross-linked polystyrene divinylbenzene copolymer resin contain-ing amino or quaternary amino groups or sulphonic acidgroups, or with a resin containing carboxyl groups, e.g. a polyacrylic acid resin. Soluble base salts (e.g. alkali metal salts such as the sodium salt) of compounds of formula (I) may be used in therapeutic applications because of the rapid distribution of such salts in the body upon administration. Where, however, insoluble salts of compounds (I) are desired in a par-ticular application, e.g. for use in depot preparations, such salts may be formed in conventional manner, for example with appropriate organic amines.
These and other salt derivatives such as the salts with toluene-~-sulphonic and methanesulphonic acids may be employed as intermediates in the prepar-ation and/or purification of the present compounds of formula (I), for example in the processes described below.
Non-toxic metabolically labile ester derivatives which may be formed from the parent compound of formu]a (I) include acyloxyalkyl esters, e.g. lower alkanoyloxy-methyl or -ethyl esters such as acetoxymethyl or -ethyl or pivaloyloxymethyl esters. In addition to the above ester derivatives, the present invention includes within its scope compounds of formula (I) in the form of other physiologically acceptable equivalents, i.e. physiologi-cally acceptable compounds which, like the metabolicallylabile esters, are converted in vivo into the parent antibiotic compound of formula (I).
Preferred compounds according to the invention ~4~320 include those compounds of formula (I) wherein Y represents a l-carbamoyl-methylpyridinium-4-yl group. Preference is also expressed for those compounds wherein Ra and Rb both represent methyl groups or together with the carbon atom to which they are attached form a cyclobutylidene group.
Particularly preferred compounds according to the invention are ~6R,7R)-7- L (z) -2-(2-aminothiazol-4-yl)-2-(2-carboxyprop-2-oxyimino)acetamido]-

3-[(1-carbamoylmethylpyridinium-4-yl)thiomethyl]ceph-3-em-4-carboxylate and (6R,7R)-7-[(Z)-2-(2-aminothiazol-4-yl)-2-(1-carboxycyclobut-1-oxyimino)acet-amido]-3-L(l-carbamoylmethylpyridinium-4-yl)thiomethyl]ceph-3-em-4-carboxylate, and their non-toxic salts and non-toxic metabolically labile esters. These particularly preferred compounds possess to an outstanding extent the general antibiotic properties set out above for the compounds of formula (I). However one may emphasise their excellent activity against strains of Pseudomonas.
The compounds also possess useful activity against strains of Staphylococcus aureus. The compounds have excellent antibacterial properties which generally are not impaired by human serum and, moreover, the effect of increased inocula against the compounds is low. The serum half-life in primates points to the probability of a comparatively long half-life in man, with the possibility of less frequent dosages being required for less serious infections. They are well distributed in the bodies of small rodents giving useful therapeutic levels after subcutaneous injection. Experimental infections in mice with gram-negative bacteria were successfully treated using the compounds and, in particular, good protection was obtained against strains of Pseudomonas aeruginosa, an organism normally not susceptible to treatment with cephalo-sporin antibiotics. The protection was comparable with the treatment with an aminoglycoside such as amikacin. Adminstration of 500 mg/kg of either compound to mice did not cause any deaths, indicating _ g _ an LD50 in excess of this figuren The above described compounds according to the invention may be used for treating a variety of diseases caused by pathogenic bacteria in human beings and animals, such as respiratory tract infections and urinary tract infections.
According to a further embodiment of the present invention we provide a process for the preparation of compounds of formula (I) as hereinbefore defined or non-toxic salts or non-toxic metabolically labile esters thereof which comprises (A) reacting a compound of the formula H H
H2N I ~f ~
O ~ N ~ CH2SY 63 (II) COO

[wherein Y ~ is as hereinbefore defined, B is > S or >S- 0 (~- or ~-) and the dotted line bridging the 2-, 3- and 4-positions indicates that the compound is a ceph-2-em or ceph-3-em compound], or an acid addition salt (formed with, for example, a mineral acid such as hydrochloric, hydrobromic, sulphuric, nitric or phosphoric acid or an organic acid such as methane-sulphonic or toluene-~-sulphonic acid) or an N-silyl derivative thereof, or a corresponding compound possessing a group of the formula -COORl at the 4-position where Rl is a hydrogen atom or a carboxyl blocking group e.g. the residue of an ester-forming aliphatic or araliphatic alcohol or an ester-forming phenol, silanol or stannanol (the said alcohol, phenol, silanol or stannanol preferably containing 1 to 20 carbon atoms) and having an associated anion A~such as a halide, e.g. chloride, bromide or iodide, or trifluoroacetate ion, with an acid of formula ~4~ZO

S N
\ - / C.CCOH (III) O.C.COOR
Rb wherein Ra and ~ are as hereinbefore defined; R2 represents a carboxyl blocking group (e.g. as described for Rl) and R3 is an amino or protected amino group or with an acylating agent corresponding thereto, or (s) reacting a compound of formNla H H
S N l , B
\ - / C.CO.NH ~ (IV) N \ ,Ra O ~ N ~ CH2X
O.C.COOR CooR4 Rb (wherein R , Rb, R3, B and the dotted line æ e as hereinbefore defined; R and R4a may independently represent hydrogen or a carboxyl blocking group; and X is a leaving group, e.g. an acetoxy or dichloroacetoxy group or a halogen atom such as chlorine, brcmlne or iodine) or a salt thereof, with a sulphur nucleophile serving to form the group -CH2SY ~3 (wherein Y ~ is as hereinbefore defined) at the 3~position; or (C) reacting a compound of the formula S N H H
\ / C.CO.NH
N Ra ~ N ~ CH2SYl (V) o.c.cooR4a 4 . COOR

$~ Rb _ 11 -1144~;~0 (wherein Ra, Rb, R3, B and the dotted lines are as hereinbefore defined; R4 and R4a in this instance are both carboxyl blocking groups and yl represents a pyridyl group) with a carbamoylmethylating agent serving to introduce a carbamoylmethyl group as a substituent on the nitrogen atom of the pyridyl ring; whereafter, if necessary and/or desired in each instance, any of the following reactions, in any appropriate sequence, are carried out:-i) conversion of a ~2-isomer into the desired ~3-isomer, ii) reduction of a compound wherein B is ,S ~0 to form a compound wherein B is ~S, iii) conversion of a carboxyl group into a non-toxic salt or non-toxic metabolically labile ester function, and iv) removal of any carboxyl blocking and/or N-protect-ing groups.
Acylating agents which may be employed in the preparation of compounds of formula (I) include acid halides, particularly acid chlorides or bromides.
Such acylating agents may be prepared by reacting an acid (III) or a salt thereof with a halogenating agent e.g. phosphorus pentachloride, thionyl chloride or oxalyl chloride.
Where an acid addition salt of the compound of formula (II) is used, this is generally treated with a base prior to reaction with the compound of formula (III) or an acylating agent corresponding thereto.
Acylations employing acid halides may be effected in aqueous and non-aqueous reaction media conveniently at temperatures of from -50 to +50C preferably -20 to +30C, if desired in the presence of an acid binding agent. Suitable reaction media include aqueous ketones such as aqueous acetone, esters such as ethyl acetate, halogenated hydrocarbons such as dichloromethane, amides such as dimethylacetamide, nitriles such as acetonitrile, or mixtures of two or more such

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solvents. Suitable acid binding agents include tertiary amines (e.g. triethylamine or dimethylaniline), inorganic bases (e.g. calcium carbonate or sodium bicarbonate), and oxiranes such as lower 1,2-alkylene oxides (e.g.
ethylene oxide or propylene oxide) which bind hydrogen halide liberated in the acylation reaction.
Acids of formula (III) may themselves be used as acylating agents in the preparation of compounds of formula (I). Acylations employing acids (III) are desirably conducted in the presence of a condensing agent, for example a carbodiimide such as N,N'-dicyclo-hexylcarbodiimide or N-ethyl-N'-~-dimethylaminopropyl-carbodiimide; a carbonyl compound such as carbonyldi-imidazole; or an isoxazolium salt such as N-ethyl-5-phenylisoxazolium perchlorate.
Acylation may be effected with other amide-forming derivatives of acids of formula (III3 such as, for example, an activated ester, a symmetrical anhydride or a mixed anhydride (e.g. formed with pivalic acid or with a haloformate such as a lower alkylhaloformate).
Mixed anhydrides may also be formed with phos-phorus acids (for example, phosphoric or phosphorous acids), sulphuric acid or aliphatic or aromatic sulphon-ic acids (for example toluene-p-sulphonic acid).
An activated ester may conveniently be formed ln situ using, for example, l-hydroxybenzotriazole in the presence of a condensing agent as set out above.
Alternatively, the activated ester may be preformed.
Acylation reactions involving the free acids or their above mentioned amide-forming derivatives are desirably effected in an anhydrous reaction medium e.g. dichloromethane, tetrahydrofuran, dimethyl-formamide or acetonitrile.
If desired, the above acylation reactions may be performed in the presence of a catalyst, e.g. 4-dimethylaminopyridine.
The amino acids of formula (III) and acylating agents corresponding thereto may, if desired, be prepared ZO

and employed in the form of their acid addition salts.
Thus, for example, acid chlorides may conveniently be employed as their hydrochloride salts and acid bromides as their hydrobromide salts.
In process (B) above, the sulphur nucleophile may be used to displace a wide variety of substituents X from the cephalosporin of formula (IV). To some extent the facility of the displacement is related to the pKa of the acid HX from which the substituent is derived. Thus, atoms or groups X derived from strong acids tend, in general, to be more easily displaced than atoms or groups derived from weaker acids. The facility of the displacement is also related, to some extent, to the precise character of the sulphur nucleo-phile. The nucleophile may be employed, for example,in the form of a thiol or thione, and may be, for example an N-carbamoylmethylpyridthione.
The displacement of X by the sulphur nucleophile may conveniently be effected by maintaining the reactants in solution or suspension. The reaction is advantageously effected using from 1 to 10 moles e.g. 1 to 2 moles of the nucleophile.
Nucleophilic displacement reactions may conven-iently be carried out on those compounds of formula (IV) wherein the substituent X is a halogen atom or an acyloxy group, for example as discussed below.

AcyloxY groups Compounds of formula (IV) wherein X is an acetoxy group are convenient starting materials for use in the nucleophilic displacement reaction with the sulphur nucleophile. Alternative starting materials in this class include compounds of formula (IV) in which X
is the residue of a substituted acetic acid e.g. chloro-acetic acid, dichloroacetic acid and trifluoroaceticacid.
Displacement reactions on compounds of formula (IV) possessing X substituents of this class, parti-cularly in the case where X is an acetoxy group, may be facilitated by the presence in the reaction medium of iodide or thiocyanate ions.
The substituent X may also be derived from formic acid, a haloformic acid such as chloroformic acid, or a carbamic acid.
When using a compound of formula (IV) in which X represents an acetoxy or substituted acetoxy group, it is generally desirable that the group R4 in formula (IV) should be a hydrogen atom and that B should represent ~S. In this case, the reaction is advantageously effected in an aqueous medium.
Under aqueous conditions, the pH value of the reaction solution is advantageously maintained in the range 6-8, if necessary by the addition of a base.
The base is conveniently an alkali metal or alkaline earth metal hydroxide or bicarbonate such as sodium hydroxide or sodium bicarbonate.
When using compounds of formula (IV) in which X is an acetoxy group, the reaction is conveniently effected at a temperature of 30C to 110C, preferably 50C to 80C.

Halogens Compounds of formula (IV) in which X is a chlor-ine, bromine or iodine atom can also be conveniently used as starting materials in the nucleophilic displace-ment reaction with the sulphur nucleophile. When using compounds of formula (IV) in this class, B may represent > S ~0 and R4 may represent a carboxyl blocking group.
The reaction is conveniently effected in a non-aqueous medium which preferably comprises one or more organic solvents, advantageously of a polar nature such as ethers, e.g. dioxan or tetrahydrofuran, esters, e.g.
ethyl acetate, amides, e.g. formamide or N,N-dimethyl-formamide or ketones e.g. acetone. Other suitable organic solvents are described in more detail in British Patent Specification No. 1,326,531. The reaction medium 1~4~ZO

should be neither extremely aciclic nor extremely basic.
In the case of reactions carried out on compounds of formula (IV) in which R4 and R4a are carboxyl blocking groups the products will be formed as the corresponding halide salts which may, if desired, be subjected to one or more ion exchange reactions to obtain salts having the desired anion.
When using compounds of formula (IV) in which X is a halogen atom as described above, the reaction is conveniently effected at a temperature of -20 to +60, preferably 0 to +30C.
In process (C) above, the 3-pyridylthiomethyl compound of formula (V) is advantageously reacted with a carbamoylmethylating agent of the formula H2NCOCH2Z
where Z is a leaving group such as a halogen atom (e.g.
iodine, chlorine or bromine) or a hydrocarbyl sulphonate (e.g. mesylate or tosylate) group. Iodoacetamide is preferred as the carbamoylmethylating agent. The reaction is preferably carried out at a temperature in the range of 0 to 60C, advantageously 20 to 30C. The reaction may be conveniently effected in an inert solvent such as an ether e.g. tetrahydrofuran, an amide, e.g. dimethyl-formamide, a lower alkanol e.g. ethanol, a lower dialkyl-ketone, e.g. acetone, a halogenated hydrocarbon e.g.
dichloromethane or chloroform, or an ester, e.g. ethyl acetate.
The 3-pyridylthiomethyl compound of formula (V) used as starting material in process (C) may be prepared for example by reaction of a compound of formula (IV) (as defined above) with an appropriate sulphur nucleophile (e.g. pyrid-4-thione) in an analogous manner to the nucleophilic displacement reaction described with respect to process (B). When X in formula (IV) is halogen, the reaction is preferably effected in the presence of an acid scavenging agent, for example a base such as triethylamine or calcium carbonate. If desired the above nucleophile may be used in the form of a metal thiolate salt.

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The reaction product may be separated from the reaction mixture, which may contain, for example, un-reacted nucleophile and other substances, by a variety of processes including recrystallisation, ionophoresis, column chromatography and use of ion-exchangers (for example by chromatography on ion-exchange resins) or macroreticular resins.
A ~2-cephalosporin ester derivative obtained in accordance with the process of the invention may be converted into the corresponding ~3 -derivative by, for example, treatment of the ~2-ester with a base such as pyridine or triethylamine.
A ceph-2-em reaction product may also be oxidised to yield the corresponding ceph-3-em l-oxide, for example by reaction with a peracid e.g. peracetic or m-chloro-perbenzoic acid; the resulting sulphoxide may, if desired, subsequently be reduced as described hereinafter to yield the corresponding ceph-3-em sulphide.
Where a compound is obtained in which B is >S- 0 this may be converted into the corresponding sulphide by, for example, reduction of the corresponding acyloxy-sulphonium salt prepared in situ by reaction with e.g.
acetyl chloride in the case of an acetoxy-sulphonium salt, reduction being effected by, for example, sodium dithionite or by iodide ions as in a solution of potassium iodide in a water miscible solvent e.g. acetic acid, acetone, tetrahydrofuran, dioxan, dimethylformamide or dimethylacetamide. The reaction may be effected at a temperature between -20 to +50C.
Metabolically labile ester derivatives of the compounds of formula (I) may be prepared by reacting a compound of formula (I) or a salt or protected deriv-ative thereof with the appropriate esterifying agent ~1~4~2V

such as an acyloxymethyl halide (e.g. iodide), convenient-ly in an inert organic solvent such as dimethylformamide or acetone, followed, where necessary, by removal of any protecting groups.
Base salts of the compounds of formula (I) may be formed by reacting an acid of formula (I) with an appropriate base. Thus, for example, sodium or potassium salts may be prepared using the respective 2-ethylhexan-oate or hydrogen carbonate salts. Acid addition salts may be prepared by reacting a compound of formula (I) or a metabolically labile ester derivative thereof with the appropriate acid.
Where a compound of formula (I) is obtained as a mixture of isomers, the syn isomer may be obtained by, for example, conventional methods such as crystalli-sation or chromatography. Syn and anti isomers may be distinguished by appropriate techniques, e.g. by thin layer, paper or high pressure liquid chromatography or by their proton magnetic resonance spectra.
For use as starting materials for the preparation of the compounds of formula (I) according to the invent-ion, compounds of general formula (III) and acid halides and anhydrides corresponding thereto in their syn isomeric form, or in the form of mixtures of the syn isomers and the corresponding anti isomers containing at least 90% of the syn isomer, are preferably used.
Acids of formula (III) (provided that Ra and Rb together with the carbon atom to which they are attached do not form a cyclopropylidene group) may be prepared by etherification of a compound of formula ~' (VI) C.COOR
N \
OH

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(wherein R3 is as hereinbefore defined and R5 represents a carboxyl blocking group) by reaction with a compound of general formula Ra T.C.COOR2 (vII) R

(wherein Ra, Rb, and R2 are as hereinbefore defined and T is halogen such as chloro, bromo, or iodo; sulphate;
or sulphonate such as tosylate) followed by removal of the carboxyl blocking group R5.
Acids of general formula (III) may also be prepared by reaction of a compound of formula \- / Co.cOOR5 (VIII) (wherein R3 and R5 are as hereinbefore defined) with a compound of formula H2~.0~C.COOR2 (IX) Rb (wherein Ra, R~ and R2 are as defined above), followed by removal of the carboxyl blocking group R5.
The last mentioned reaction is particularly applic-able to the preparation of acids of formula (III) wherein Ra and Rb together with the carbon atom to which they are attached form a cyclopropylidene group.
These methods of preparing the acids are described in more detail in Belgian Patent Specification No. 876538.
The acids of formula (III) may be converted into ~1~4C32~

the corresponding acid halides and anhydrides and acid addition salts by conventional methods Where X is a halogen (i.e. chlorine, bromine or iodine) atom in formula (IV), ceph-3-em starting compounds may be prepared in conventional manner, e.g.
by halogenation of a 73-protected amino-3-methylceph-3-em-4-carboxylic acid ester l~-oxide, removal of the 7~ protecting group, acylation of the resulting 7~-amino compound to form the desired 7~-acylamido group, e.g. in an analogous manner to process (A) above, followed by reduction of the l~-oxide group later in the sequence.
This is described in British Patent No. 1,326,531.
The corresponding ceph-2-em compounds may be prepared by the method of Dutch published Patent Application No. 6,902,013 for example by reaction of a 3-methylceph-2-em compound with N-bromosuccinimide to yield the corresponding 3-bromomethylceph-2-em compound.
Where X in formula (IV) is an acetoxy group, such starting materials may be prepared for example by acylation of 7-aminocephalosporanic acid, e.g.
in an analogous manner to process (A) above. Compounds of formula (IV) in which X represents other acyloxy groups can be prepared by acylation of the correspon-ding 3-hydroxymethyl compounds which may be prepared for example by hydrolysis of the appropriate 3-acetoxy-methyl compounds e.g. as described inter alia in British Patent Specifications 1,474,519 and 1,531,212.
Compounds of formula (II) may likewise be prepared in conventional manner, e.g. by nucleophilic displace-ment of a corresponding 3-acyloxymethyl or 3-halomethyl compound with an N-carbamoylmethylpyridthione.
A further method for the preparation of starting materials of formula (II) comprises deprotecting the corresponding protected 7~-amino compound in conventional manner, e.g. using POC13 in methanol or PC15.
It is to be noted that compounds of formula (II) are novel and constitute a further aspect of the present invention.

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The sulphur nucleophile used in process (B) above may be prepared by reacting a bromopyridine, e.g. 4-bromopyridine with iodoacetamide followed by a sulphide or hydrosulphide salt, e.g. sodium sulphide, to give the l-carbamoylmethylpyridthione, e.g. l-carbamoyl-methylpyrid-4-thione. These sulphur nucleophiles are novel and constitute a still further aspect of the present inventionn It should be appreciated that in some of the above transformations it may be necessary to protect any sensitive groups in the molecule of the compound in question to avoid undesirable side-reactions. For example, during any of the reaction sequences referred to above it may be necessary to protect the NH2 group of the aminothiazolyl moiety, for example by tritylation, acylation (e.g. chloroacetylation), protonation or other conventional method. The protecting group may thereafter be removed in any convenient way which does not cause breakdown of the desired compound, e.g. in the case of a trityl group by using an optionally halogen-ated carboxylic acid such as acetic acid, formic acid, chloroacetic acid or trifluoroacetic acid or using a mineral acid, e.g. hydrochloric acid or mixtures of such acids, conveniently in the presence of a protic solvent such as water or, in the case of a chloroacetyl group, by treatment with thiourea.
Carboxyl blocking groups used in the preparation of the compounds of formula (I) or in the preparation of necessary starting materials are desirably groups which may readily be split off at a suitable stage in the reaction sequence, conveniently at the last stage. It may, however, be convenient in some instances to employ biologically acceptable, metabolically labile carboxyl blocking groups such as acyloxy-methyl or -ethyl groups (e.g. acetoxy-methyl or -ethyl or pivaloyloxy-methyl groups) and retain these in the final product to give a biologically acceptable ester derivative of the compound of formula (I).

-Suitable carboxyl blocking groups are well known in the art, a list of representative blocked carboxyl groups being included in British Patent No. 1,399,086.
Preferred blocked carboxyl groups include aryl lower alkoxycarbonyl groups such as p-methoxybenzyloxycarbonyl, ~-nitrobenzyloxycarbonyl and diphenylmethoxycarbonyl;
t-butoxycarbonyl; and lower haloalkoxycarbonyl groups such as 2,2,2-trichloroethoxycarbonyl. Carboxyl blocking group(s) may subsequently be removed by any of the appropriate methods disclosed in the literature; thus, for example, acid or base catalysed hydrolysis is applic-able in many cases, as are enzymically-catalysed hydro-lyses.
The following Examples illustrate the invention.
All temperatures are in C. "Ether" refers to diethyl ether, "petrol" refers to petroleum ether (b.p. 40-60) "Kieselgel" is chromatographic silica and "Calofort U" is a form of finely divided calcium carbonate. Proton magnetic resonance spectra were determined on the products at 100 MHz. The integrals were in agreement with the assignments; the signs of the coupling constants, J, in Hz, were not determined. The following abbreviations are used:
s = singlet, d = doublet, m = multiplet, br = broad and ABq = AB-quartet.

11~4~Z10 Preparation 1 4-Bromo~l-Carbamo~rlmethylpyridinium Iodide A mixture of 4-bromopyridine hydrochloride (980 mg.), water (5 ml.), ether (10 ml.) and potassium hydroxide (400 mg.) was shaken and the separated aqueous layer was extracted with more ether (10 ml.). The combined organic solution was dried ~Na2SO4) and treated with a solution of iodoacetamide (1.4 9.) in acetone (15 ml.). The mixture was left to stand in the dark at 22 for 3 days. The crystals obtained were filtered off, washed with ether and dried in vacuo to give the title compound (308 mg.), I (DMSO-d6) 1.04 (d, J 7Hz) (pyrid-2 and 6-yl), 1.43 (d, J 7Hz) (pyrid-3 and 5-yl), 1.88 and 2.27 (2 br.s.) (CONH2), 4.52 (5, N~CH2 ) .
Preparation 2 l-(Carbamoylmethyl)pyrid-4 thion 4-Bromo-l-carbamoylmethylpyridinium iodide (810 mg.) in ethanol (20 ml.) was treated with anhydrous sodium sulphide (205 mg.) and the mixture was stirred and refluxed for 30 minutes. The mixture was left to cool and was then filtered. The filtrate was-treated with ether (60 ml.) and the resulting precipitate was filtered off, washed with ether and dried rapidly in vacuo to give a solid which was purified on a column of silica gel eluted with chloroform: ethanol 1:1.
50 ml. fractions were collected and fractions 4 to 7 were combined and evaporated down to about 10 ml.
The mixture was heated briefly to boiling and was then left to cool at 4 overnight. The crystals were filtered off and dried in vacuo to give the title comPound (63 mg.), m.p. (Kofler) 260 to 264 (decomp.), ~max ( lcm 356)~ ~max 351-5 nm (E1Cm 1 inf 293 nm (Eicm 80) (EtOH).

~14~ZV

Example 1 a) DiPhenylmethyl (lS,6R,7R)-7-[(Z)-2-t2-t-Butoxy-carbonylprop-2-oxyimino~-2-(2-tritylaminothiazol-4-yl)acetamido]-3-(pyrid-4-ylthiomethyl)ceph-3-em-4-car-boxylate, l-Oxide.
A stirred mixture of diphenylmethyl (lS,6R,7R)-3-bromomethyl-7-[(Z)-2-(2-t-butoxycarbonylprop-2-oxyimino)-2-(2-tritylaminothiazol-4-yl)acetamido~ceph-3-em-4-carboxylate,l-oxide (1.54 g) and 4-mercaptopyridine (0.195 g) in dry tetrahydrofuran (12 ml) was treated with triethylamine (0.224 ml) to give a red mixture.
After stirring vigorously at 22 for 3 hours, the resultant product was partitioned between ethyl acetate and water (containing a little brine). The organic layer was washed with water (twice) and dried and evaporated in vacuo to give a foam (1.647 9).
This foam was purified by chromatography on a column of Merck~Kieselgel~60 (70 to 230 mesh, 80 9) which was eluted with toluene:ethyl acetate (3:2) in 80 ml fractions. Appropriate fractions were collected, combined and evaporated to give the title compound (0-92 9) as a foam; [~]D -7 (c, 0.88, CHC13), ~maX(EtOH) 255 nm (ElCm252).

b) Diphenylmethyl (lS,6R,7R)-7-[(Z)-2-(2-t-Butoxy-carbonylprop-2-oxyimino)-2-(2-tritylaminothiazol-4-yl)acetamido]-3-[(1-carbamoylmethylpyridinium-4-yl)thio-methyl]ceph-3-em-4-carboxylate, l-oxide, Iodide Salt The product of stage (a) (0.106 g) and iodoacetamide (0.055 g) were suspended in chloroform (0.5 ml) and left to stand at 22 for 18 hours. The mixture was then stirred at 22 for 6 hours and left to stand at ca. 15 for 21 days. The mixture was diluted with ethyl acetate (ca. 3 ml) and added dropwise to ether (25 ml). The precipitate was filtered off and washed with ether and dried ln vacuo to give the title compound (0.105 g) as a solid; [~]D-14 (c, 0.28, CHC13) ~max(CHBr3) ~r~ ~ ~i76~

~4~320 _ 25 -3550 to 2800 (NH and NH2), 1806 (~-lactam), 1735 (CO2R), 1690 and 1518 (CONH), 1638 (C=N), 1604 and 1499 (C=C, aromatic) and 755 cm 1 (phenyl).

c) Diphenylmethyl (6R,7R)-7-[(Z)-2-(2-t-ButoxycarbonYl-~_op-2-oxyimino)-2-(2-tritylaminothiazol-4-yl)acetamido]-3-[(1-carbamoylmethylpyridinium-4-yl)thiomethyl]-ceph-3-em-4-carboxylate, Iodide Salt The product of Stage (b) (0.744g) in acetone (5 ml) was treated with dry powdered potassium iodide (0.4 g).
The stirred and cooled (-10) mixture was treated with acetyl chloride (0.086 ml) and the product was stirred at 0 to 2 for 1 1/4 hours. The mixture was added slowly to a stirred solution of sodium metabisulphite (1 g) in water (100 ml) and the precipitate was filtered off and washed with water and dried in vacuo over phos-phorus pentoxide to give a solid (0.679 g) containing some starting material.
The reduction sequence was repeated on the product (0.679 g) exactly as described above (except that reaction time at 0 to 2 was 50 minutes) to give a precipitate which was extracted into chloroform. The organic solution was washed with water and dried and evaporated to give the title compound (0.636 g) as a foam, [~]D-37C (c, 3 ' max (EtOH) 307.5 nm (El% 170) with an inflection at 260 nm (ElCml63) and vmax(CHBr3) 3460, 3406 and ca. 3250 (NH and NH2), 1800 (~-lactam), 1730 to 1690 (broad, CO2R and CONH), 1605 and 1500 (C=C, aromatic) 1528 (CONH) and 760 cm 1 (phenyl).

d) ~6R,7R)-7-[(Z)-2-(2-Aminothiazol-4-Yl)-2-(2-carboxy-prop-2-oxyimino)acetamido]-3-[(1-carbamoylmethylpyridinium-4-yl)thiomethyl]ceph-3-em-4-carboxylate The product of stage (c) (0.53 g) was treated with anisole (1 ml) and trifluoroacetic acid (4 ml) was added thereto. The suspension was swirled for 2 minutes at 22 and was then evaporated to an oil which was triturated with ether and dried ln vacuo 4~3ZO
_ 26 -to give a solid (0.392 g) which was treated with anisole (0.12 ml) followed by trifluoroacetic acid (15 ml).
The suspension was swirled occasionally at 22 over 15 minutes and was then filtered. The residue was washed with trifluoroacetic acid (2x5 ml) and the filtrate and washings were evaporated to an oil which on trituration with ether gave a solid which was filtered off, washed with ether and dried ln vacuo to give the title comPound (0.352 9) as a solid associated with 1.1 moles of tri-fluoroacetic acid; [~]D-50 (e 0.35, DMSO), ~max (pH6 buffer) 232 nm (E1Cm 283) and 308 nm (ElCm 317) with an inflection at 260 nm (Elcm 204)-Exa~ple 2 a) DiphenylmethYl (lS,6R,7R)-7-[(Z)-2-(1-t-butoxy-earbonylcyclobut-1-oxyimino)-2-(2-tritYlaminothiazol-carboxylate, l-oxide Diphenylmethyl (lS,6R,7R)-3-bromomethyl-7-[(Z)-2-(1-t-butoxyearbonyleyelobut-1-oxyimino)-2-(2-tritylamino-thiazol-4-yl)aeetamido]eeph-3-em-4-earboxylate, l-oxide (4.86 9) in aeetone (150 ml) was treated with 4-mereapto-pyridine (693 mg) and Calofort U~(1.56 9). The mixture obtained was refluxed for 80 minutes, filtered and the residue washed with aeetone. The filtrate and washings were evaporated to give a solid which was dissolved in dichloromethane (100 ml) and washed with saturated aqueous sodium hydrogen carbonate (100 ml), water (100 ml), brine (100 ml) and dried over anhydrous sodium sulphate. The solvents were evaporated to give a foam. This was ehromatographed on a column of Kieselgel 63 (160 9) and the column eluted with dichloromethane:acetone (3:1). Appropriate fractions were combined and evaporated to give the title compound as a foam (2.98 9), vmax (CHBr3) 3400 (NH), 1806 (~-laetam), 1722 (CO2R), 1680 and 1514 em 1 (CONH) and T (CDC13) values include 1.74 and 3.08 (2 doublets; pyridyl protons), 3.00 (s; CHPh2), 3.28 (s; thiazolyl proton), 3.88 (dd, J10 and 5; 7-~r~ ~ ~r~

_ 27 _ H), 5.48 (d, J5; 6-H), 5.45 and 6.23 (2 doublets; 3-CH2), 7.46 and 8.0 (multiplets; cyclobutyl protons), 8.55 (s; t-butyl group).

b) Diphenylmethyl (lS,6R,7R)-7-[(Z)-2-(1-t-butoxy-carbonylcyclobut-1-oxyimino)-2-(2-tritylaminothiazol-4-yl)acetamido]-3-[(1-carbamoylmethylpyridinium-4-yl)thio-methyl]ceph-3-em-4-carboxylate, l-oxide, Iodide salt The product of Stage a) (2.9 g) and iodoacetamide (0.99 g) were dissolved in chloroform (16 ml) and stirred 10 at 21 for 16 hours and then stood at 22 for 3 days.
The solution was then added dropwise to stirred ether (500 ml) and filtered to give the title comPound as a solid (3-08 g), vmax (Nujol~ 3340 (NH and NH2), 1800 -lactam), 1724 (CO2R), 1690 and 1520 (CONH) and 1690 cm 2)' ~max (EtOH) 309 nm (ElCm 101).

c) Diphenylmethyl (6R,7R)-7-[(Z)-l-t-butoxycarbonyl-cyclobut-l-oxyimino)-2-(2-tritylaminothiazol-4-yl)acetamido]-3-[(1-carbamoylmethylpyridinium-4-yl)thiomethyl]ceph-3-em-4-carboxylate, Iodide salt The product of Stage b) (2.9 g) in acetone (20 ml) was treated with potassium iodide (1.54 g) and the mixture cooled to -10 and treated with acetyl chloride (0.33 ml). The mixture was stirred at 0 to 5 for 21 hours and then slowly added to a stirred solution of sodium metabisulphite (4 g) in water (10 ml) and the precipitate filtered off, washed with water and dried over phosphorus pentoxide to give the title compound (2.52 9)~ ~max (EtOH) 308 nm (ElCml57), vmax (CHBr3) 3460, 3400, 3260 (NH and NH2), 1794 (~-lactam), 1720 (CO2R), 1700 (CONH2) and 1690 and 1526 cm 1 (CONH).

d) (6R,7R)-7-[(Z)-2-(2-Aminothiazol-4-yl)-2-(1-carboxy-cyclobut-l-oxyimino)acetamido]-3-[(1-carbamoylmethyl-pyridinium-4-yl)thiomethyl]ceph-3-em-4-carboxylate The product of Stage c) (2.05 g) was treated with anisole (4 ml) and trifluoroacetic acid (16 ml).
.. ~

The suspension was swirled at 22 for 2 minutes and the solvents evaporated to give an oil which was triturated with ether to give a solid. This solid was suspended in anisole (2.5 ml) and trifluoroacetic acid (58 ml) and swirled for 15 minutes at 22 and then filtered.
The residue was washed with trifluoroacetic acid (2 x 5ml) and the filtrate washings evaporated to give an oil which on trituration with ether gave a solid. This was dissolved in trifluoroacetic acid (40 ml) and water (100 ml) was added. The solution was stirred for 30 minutes at 22, concentrated to ca 60 ml in vacuo and washed with ether (3 x 100 ml). The aqueous phase was then briefly evaporated and freeze-dried to give the title_compound as a solid (1.13 9) associated with 1.5 moles of trifluoroacetic acid, ~max (EtOH) 236, 260 and 309.5 nm (ElCm 244, 167 and 257), vmax (Nujol) 3300 (NH2 and NH), 1780 (~-lactam), 1670 (CF3CO2), 2600 and 1720 (CO2H), 1710 (CONH2), 1690 and 1550 cm (CONH). The product was further purified by high pressure liquid chromatography and column chromatography.

'3~
.

Example 3 a) Diphenylmethyl (6R,7R)-7-[(Z)-2-(1-t-butoxycarbonYl-cyclobut-l-oxyimino)-2-(2-tritylaminothiazol-4-yl)acetamido]-3-[(1-carbamoylmethylpyridinium-4-yl)thiomethyl]ceph-3-em-4-carboxylate, Bromide Diphenylmethyl (6R,7R)-3-bromomethyl-7-[(Z)-2-(l-t-butoxycarbonylcyclobut-l-oxyimino)-2-(2-tritylamino-thiazol-4-yl)acetamido]ceph-3-em-4-carboxylate (300 mg.) in dry tetrahydrofuran (5 ml.) was treated with 1-carbamoylmethylpyrid-4-thione (53 mg.) and the suspension obtained was stirred at 22 for 24 hours. The reaction mixture was diluted with chloroform (10 ml.), filtered and the filtrate was evaporated down to about 2 ml., then diluted with petrol (10 ml.) The fine precipitate was filtered off and dried in vacuo to give the title com~o~nd (284 mg.) as a solid, []D ~ 54 (c 0.47, C~C13), ~max 310 nm (Elcm 197), ~inf 236 nm (ElCm 250 ), ~inf 258 nm (ElCm 175) (EtOH) b) (6R,7R)-7-[(Z)-2-(2-Aminothiazol-4-yl)-2-(1-carboxy-cyclobut-1-oxvimino)acetamido]-3-[(1-carbamoylmethyl-pyridinium-4-yl)thiomethyl]ceph-3-em-4-carboxylate.
The above product from stage (a) (1.17 9) was treated with anisole (1.2 ml.) and trifluoroacetic acid (4.8 ml.) and the mixture obtained was swirled at 22 for 5 minutes until the solid had dissolved.
The solution was evaporated to an oil which was triturated with ether to give a solid. The solid was moistened with anisole (0.12 ml.) and treated with trifluoroacetic acid (12 ml.). The resultant solution was left to stand at 22 for 15 minutes and was then evaporated to an oil which was triturated with ether (50 ml.).
The solid was filtered off, washed with ether and dried in vacuo. The residue was treated with warm (30) trifluoroacetic acid:water = 1:4 (50 ml.) and the faint suspension was swirled at 30 for 10 minutes and was then washed with ethyl acetate (50 ml.). The clear aqueous layer was washed with ether (50 ml.) and then 3~20 concentrated by evaporation and freeze dried to give the title compound associated with trifluoroacetic acid (375 mg.) as a foam [~]D -14 (c 0.52, H2O:EtOH
= 1:1), ~ 235 nm (E1cm 250)~ ~max lcm 329)~ ~inf 255 nm (Elcm 188) (pH 6-4 buffer)-Example 4 (6R,7R)-7-[(Z)-2-(2-Aminothiazol-4-yl)-2-(1-carboxy-cyclobut-l-oxyimino)acetamido]-3-[(1-carbamoylmethyl-pyridinium-4-yl)thiomethyl]ceph-3-em-4-carboxylate sodium salt.
A mixture of (6R,7_)-3-acetoxymethyl-7-~(Z)-2-(aminothiazol 4- yl)-2-(1-carboxycyclobut-1-oxyimino)-acetamido]ceph-3-em-4-carboxylic acid hydrate (1.165 15 g), 1-(carbamoylmethyl)pyrid-4-thione (697 mg) and sodium bicarbonate (659 mg) in water (2 ml) was stirred at 80C under nitrogen for 1.1/4 h. The solution obtained was cooled, diluted with water and then added dropwise with stirring to acetone (600 ml) to give a solid.
A solution of this solid in water ~30 ml) was chromatographed on a column of Amberlite XAD-2 resin.
The column was eluted with water (1400 ml) followed by water:ethanol (3:1; 800 ml), 200 ml fractions being taken. Fractions 6 - 11 were combined, concentrated by evaporation and freeze dried to give the title comPound as a foam (525 mg), []D ~9 (c 0.94, water), ~max (pH 6.4 buffer) 235.5 nm (E~Cm 304) and 309 nm (ElCm 295~, inflection at 250 nm (ElCm 255).
Example 5 (6R,7R)-7-[(Z)-2-(2-Aminothiazol-4-yl)-2-(2-carboxy-prop-2-oxyimino)acetamido]-3-[(l-carbamoylmethylpyridinium 4-yl)thiomethyl]ceph-3-em-4-carboxYlate, sodium salt.
(6R,7R)-3-Acetoxymethyl-7-[(Z)-2-(2-aminothiazol~
4-yl)-2-(2-carboxyprop-2-oxyimino)acetamido]ceph-3-em-4-carboxylic acid hydrochloride (564 mg), sodium bicarbonate (275 mg), water (1 ml), l-(carbamoylmethyl)-pyrid-4-thione (335 mg) and sodium iodide (1.8 g) were /e ~ Jk-1144~3ZID

heated together at 80 for 1.1/4 hours. The solution was allowed to cool to room temperature, water (1 ml) was added and the solution added dropwise to stirred acetone (250 ml). The precipitate obtained was filtered off, washed with acetone and ether and dried in vacuo.
The above solid was purified on a column of Amberlite XAD-2 (60 g) eluting first with water (7x60 ml fractions) and then water-ethanol 3:1 (5x60 ml fractions). The latter fractions were combined and concentrated by evaporation and finally freeze dried to give the title comPound as a foam (253 mg) [a]D -14 (c 0.5, H20), ~max 234-5 nm (Elcm 338), ~max 309 nm (ElCm 338), ~inf 254 nm (ElCm 249) (pH 6 buffer)-15Example 6 a) Diphenylmethyl (lS,6R,7R)-7-formamido-3-(pYrid-4-yl)thiomethylceph-3-em-4-carboxylate l-oxide, Iodide Diphenylmethyl (lS,6R,7R)-3-bromomethyl-7-formamido-ceph-3-em-4-carboxylate (3.03 g) in acetone (100 ml) was treated with 4-mercaptopyridine (1.0 9) and "Calofort U" (1.2 g). The mixture was stirred and refluxed under nitrogen for 20 minutes, and it was then filtered.
The filtrate was evaporated to a foam. The residue from the filtration was shaken with chloroform (250 ml) and water (150 ml), was filtered again and the filtrate was added to the foam. The aqueous phase of the mixture was treated with sodium bicarbonate solution until it was at pH 7. A little sodium chloride solution was added to clear the emulsion, then the organic phase was separated, washed, (water, 100 ml) dried (Na2SO4) and evaporated to a crystalline solid. The solid was triturated with ether (ca 100 ml) and then filtered off and dried in vacuo to give the title compound (2.68 9), [1]D ~3 (c 0.93, CHC13), m.p. (Kofler) 152 to 156 (with decomp.) b) Diphenylmethyl (lS,6R,7R)-3-[(1-carbamoylmethyl-pyridinium-4-yl)thiomethyl]-7-formamidoceph-3-em-4-_ .

\20 carboxylate, l-oxide, iodide The product from stage a) (2.4 g) in N,N-dimethyl-formamide (12 ml) was treated with iodoacetamide (1.6 g) and the solution obtained was left to stand in the dark at 22 for 30 hours. The solution was added slowly to stirred ethyl acetate (250 ml) and the precipitate thus formed was filtered off and washed with ethyl acetate (2 x 75 ml) and ether (50 ml). The solid was then dried in vacuo to give the title com~ound (3.13 g) as a solid, [~]~ -54 (c 0.47, CHC13, ~max 311 nm (Elcm 118), ~ 266 n~ (Elcml04)~ ~max 457 nlm% (ElCm39 )~ inf 251 nm (ElCml07), ~inf 257 nm (Elcm 10 ) ( c) Diphenylmethyl (lS,6R,7R)-7-amino-3-[(1-carbamoyl-methylpyridinium-4-yl)thiomethyl]ceph-3-em-4-carboxYlate, l-oxide, Iodide, Hydrochloride The product from stage b) (5.74 g), in methanol (80 ml) was stirred at 0 and to the suspension obtained was added phosphoryl chloride (2.5 ml) dropwise over 10 minutes. The mixture was stirred vigorously at 0 for 2 hours and was then diluted with ether (200 ml).
The solid obtained was filtered off and washed with ether (3 x 50 ml) then dried in vacuo. This solid was resuspended in methanol (80 ml) and treated with phosphoryl chloride (2.5 ml) as above, with the same work-up to give a solid (4.75 g). The solid was triturated with ether (100 ml), filtered off, washed with ether (2 x 50 ml) and dried in vacuo to give the title compound (4-50 g)~ [a]D +18 (c 0-32, H20), ~max 310i%5 nm (Elcm )~ inf 2741%nm (Elcml24)r ~inf 234 nm (Elcml73)~
inf 262 nm (Elcmlo7) (EtOH).

d) Diphenylmethyl ~lS,6R7R)~7-[(z)-2-(1-t-butoxycarbonyl-cyclobut-l-oxyimino)-2-(2-tritylaminothiazol-4-yl)-acetamido]-3-[(1-carbamoylmethylpyridinium-4-yl)thio-methyl]ceph-3-em-4-carboxylate, l-oxide, iodide A solution of (Z)-2-(1-t-butoxycarbonylcyclobut-. ~

11~4~:0 l-oxyimino)-2-(2-tritylaminothiazol-4-yl)acetic acid (583 mg) in dry tetrahydrofuran (7 ml) was treated with l-hydroxybenztriazole monohydrate (148 mg) and N,N'-dicyclohexylcarbodiimide (247 mg) and the mixture was stirred at 22 for 30 minutes.
The product from stage (c) (730 mg) was dissolved in N,N-dimethylformamide (20 ml) and propylene oxide (0.1 ml) was added thereto. The tetrahydrofuran mixture was filtered into the dimethylformamide solution.
The resulting solution was treated with more propylene oxide (0.1 ml) and was then stirred at 22 for 2 hours and finally left to stand at 22 for a further 16 hours.
The solution was concentrated slightly by evaporation and was partitioned between cnloroform (150 ml) and lS lN hydrochloric acid (150 ml). The organic layer was washed with 2N hydrochloric acid, water and brine and was then dried (Na2SO4) and evaporated to an oil, which was added slowly to stirred ether (150 ml). The resulting precipitate was filtered off, washed with ether and dried in vacuo to give the title compound (650 mg) as a solid [a]D -111 (c 0.34, C~C13). The sample was identical with the product of Example 2(b) by HPLC.

e) Diphenylmethyl (6R,7R)-7-[(Z)-2-(1-t-butoxycarbonyl-cyclobut-l-oxyimino)-2-(2-tritylaminothiazol-4-yl) acetamido]-3-[(1-carbamoylmethylpyridinium-4-yl)thio-methyl]ceph-3-em-4-carboxylate, iodide A solution of the product of stage d) (503 mg) in dry N,N-dimethylformamide (3 ml) was treated with dry potassium iodide (330 mg) and the solution was cooled to -10 then treated with acetyl chloride (0.075 ml).
The mixture was stirred at 0 to 5 for 1 hour and was then added slowly to a stirred solution of sodium metabisulphite (0.5 g) in water (50 ml). The precipitate obtained was filtered off, washed with water and then partitioned between chloroform (100 ml) and dilute sodium metabisulphite solution (100 ml). The organic layer was washed with water and brine then dried (Na2SO

and evaporated to a glass which was dissolved in chloro-form (3 ml). The solution was added slowly to stirred petrol (75 ml) and the precipitate obtained was filtered off and washed with petrol, then dried in vacuo to give a solid.
The solid was dissolved in acetone (2 ml) and treated with dry powdered potassium iodide (220 mg) and acetyl chloride (0.05 ml), stirred for one hour, then diluted with a so]ution of sodium metabisulphite (0.5 g) in water (50 ml) and worked up as described above to give the title compound (285 mg) as a solid, [a] -95 (c 0.29, CHC13), ~max 312 nm (Elcm )' inf 260 nm (ElCm 189) (CHC13).

f) (6R,7R)-7-[(Z)-2-(2-Aminothiazol~4-yl)-2-(1-carboxy-cyclobu~ oxyimino)acetamido]-3-[(l-carbamoylmethy pyridinium-4-yl)thiomethyl]ceph-3-em-4-carboxylate , The product from stage e) (220 mg) was treated with anisole (0.25 ml) and then with trifluoroacetic acid (1 ml) and worked-up substantially as described in Example 3b) to give the title compound associated with trifluoroacetic acid (52 mg) as a foam, ~max 233nm ~ m ). max 308.5 nm (Elcm259)l ~i f 254 nm (ElCml92) (pH 6 buffer). The NMR spectrum resembled that of the product of Example 3b).

PHARMACEUTICAL FORMULATIONS

The antibiotic compounds of the invention may be formulated for administration in any convenient way, by analogy with other antibiotics and the invention therefore includes within its scope pharmaceutical compositions comprising an antibiotic compound in accor-dance with the invention adapted for use in human or veterinary medicine. Such compositions may be presented for use in conventional manner with the aid of any necessary pharmaceutical carriers or excipients.
The antibiotic compounds according to the inven~
tion may be formulated for injection and may be pres-ented in unit dose form in ampoules, or in multi-dose lS containers if necessary with an added preservative.
The compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilising and/or dispersing agents. Alternatively, the active ingredient may be in powder form for cons-titution with a suitable vehicle, e.g. sterile, pyrogen-free water, before use.
If desired, such powder formulations may contain an appropriate non-toxic base in order to improve the water-solubility of the active ingredient and/or to ensure that when the powder is constituted with water, the pH of the resulting aqueous formulation is physio-logically acceptable. Alternatively the base may be present in the water with which the powder is constituted.
The base may be for example an inorganic base such as sodium carbonate, sodium bicarbonate or sodium acetate or an organic base such as lysine or lysine acetate.
The antibiotic compounds may also be formulated as suppositories e.g. containing conventional suppos-itory bases such as cocoa butter or other glycerides.
For medication of the eyes or ears, the prepar-ations may be formulated as individual capsules, in 32~

liquid or semi-solid form, or as drops.
Compositions for veterinary medicine may also, for example, be formulated as intramammary preparations in either long acting or quick-release bases.
The compositions may contain from 0.1% upwards, e.g. 0.1-~9% of the active material, depending on the method of administration. When the compositions comprise dosage units, each unit will preferably contain S0-1500 mg of the active ingredient. The dosage as employed for adult human treatment preferably ranges from 250 to 6000 mg per day, depending on the route and frequency of administration. For example, in adult human treatment 1000 to 3000 mg per day administered intravenously or intramuscularly should normally suffice. In treating Pseudomonas infections higher daily doses may be required.
The antibiotic compounds according to the inven-tion may be administered in combination with other therapeutic agents such as antibiotics, for example penicillins or ether cephalosporins.
The following formulations illustrate how the compounds according to the invention may be made up into pharmaceutical formulations.

EXAMPLE A
Formulation for Injection Fill sterile (6R,7R)-7-[(Z)-2-(2-aminothiazol-4-yl)-2-(carboxyprop-2-oxyimino)acetamido]-3-[(1-carbamoyl-methylpyridinium-4-yl)thiomethyl]ceph-3-em-4-carboxylate monosodium salt into glass vials such that each vial contains an amount equivalent to 500 mg of the antibiotic acid. Carry out the filling aseptically under a blanket of sterile nitrogen. Close the vials using rubber discs, or plugs, held in position by aluminium overseals thereby preventing gaseous exchange or ingress of micro-organisms. Constitute the product by dissolving in Water for Injections or other suitable sterile vehicle shortly before administration.

EXAMPLE B
Formulation for Injection A similar formulation may be prepared as in Example 20 A using (6R,7R)-7-[(Z)-2-(2-aminothiazol-4-yl)-2-(1-carboxycyclobut-l-oxyimino)acetamido]-3-[(1-carbamoylmethyl-pyridinium-4-yl)thiomethyl]ceph-3-em-4-carboxylate, monosodium salt; such that each vial contains an amount e~uivalent to 1.0 g of the antibiotic acid.
EXAMPLE C
Formulation for Injection Formula Per vial (6R,7R)-7-[(Z)-2-(2-Aminothiazol-4-yl)-2-(2-carboxyprop-2-oxyimino)acetamido]-3-[(1-carbamoylmethylpyridinium-4-yl)thiomethyl]ceph-3-em-4-carboxylate ....1.00 g.

Sodium carbonate, anhydrous ................ 100 mg METHOD
Blend the sterile cephalosporin antibiotic with sterile sodium carbonate under aseptic conditions.

~4~20 Fill aseptically into glass vials under a blanket of sterile nitrogen. Close the vials using rubber discs or plugs, held in position by aluminium overseals, thereby preventing gaseous exchange or ingress of micro-organisms. Constitute the product by dissolving in Water for Injections or other suitable sterile vehicle shortly before administration.

EXAMPLE D
Formulations for Injection Formula per vial (6R,7R)-7-[(i)-2-(2-Aminothiazol-4-yl)-2-(1-carboxy-cyclobut-l-oxyimino)acetamido]-3-[(1-carbamoylmethyl-pyridinium-4-yl)thiomethyl]ceph-3-em-4-carboxylate 500 mg.

Lysine Acetate..................................... l91 mg.

METHOD
As in Example C.

Claims (9)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A process for the preparation of a cephalosporin antibiotic of formula I

( I ) (wherein Ra and Rb, which may be the same or different, each represents a C1-2 alkyl group, or together with the carbon atom to which they are attached form a C3-6 cycloalkylidene group and Y? represents an N-carbamoylmethylpyridinium group) and non-toxic salts and non-toxic metabolically labile esters thereof, which process comprises (A) acylating a compound of the formula (II) [wherein Y? is as defined above, B is >S or >S-O (.alpha.- or .beta.-) and the dotted line bridging the 2-, 3- and 4-positions indicates that the compound is a ceph-2-em or ceph-3-em compound], or an acid addition salt or an N-silyl derivative thereof, or a corresponding compound possessing a group of formula -COOR1 at the 4-position (where R1 is a hydrogen atom or carboxyl blocking group) and having an associated anion A?, with an acid of formula (III) [wherein Ra and Rb are as defined above; R represents a carboxyl blocking group; and R3 is an amino or protected amino group] or with an acylating agent corresponding thereto, (B) reacting a compound of formula (IV) (wherein Ra, Rb, R3, B and the dotted line are as defined above;

R4 and R4a may independently represent hydrogen or a carboxyl blocking group; and X is a leaving group or salt thereof, with a sulphur nucleophile serving to form the group -CH2SY? (wherein Y?
is as defined above) at the 3-position; or (C) reacting a compound of the formula (V) (wherein Ra, Rb, R3, B and the dotted lines are as defined above;
R4 and R4a in this instance are both carboxyl blocking groups and y1 represents a pyridyl group) with a carbamoylmethylating agent serving to introduce a carbamoylmethyl group as a sub-stituent on the nitrogen atom of the pyridyl nucleus; whereafter, if necessary and/or required in each instance, any of the follow-ing reactions in any appropriate sequence, are carried out:
(i) conversion of a .DELTA.2-isomer into the desired .DELTA.3-isomer, (ii) reduction of a compound wherein B is >S-O to form a compound wherein B is >S, (iii) conversion of a carboxyl group into a non-toxic salt or non-toxic metabolically labile ester function, (iv) removal of any carboxyl blocking and/or N-protecting groups.

2. A process as claimed in claim 1 wherein Y? represents an N-carbamoylmethylpyridinium-4-yl group.

3. A compound of formula I as defined in claim 1 or a non-toxic salt or a non-toxic metabolically labile ester thereof when prepared by a process according to claim 1 or an obvious chemical equivalent thereof.

4. A process according to claim 1 wherein Y? represents an N-carbamoylmethylpyridinium-4-yl group and R and R are both methyl groups.

5. A process for preparing (6R, 7R)-7-[(Z)-2-(2-amino-thiazol-4-yl)-2-(2-carboxyprop-2-oxyimino)acetamido]-3-[(1-carbamoylmethylpyridinium-4-yl)thiomethyl]ceph-3-em-4-carboxylate, sodium salt which comprises reacting (6R, 7R)-3-acetoxymethyl-7-[(Z)-2-(2-aminothiazol-4-yl)-2-(2-carboxyprop-2-oxyimino)acetamido]-ceph-3-em-4-carboxylic acid hydrochloride with l-(carbamoylmethyl)-pyrid-4-thione in the presence of sodium bicarbonate.

6. The compound(6R, 7R)-7-[(Z)-2-(2-aminothiazol-4-yl)-2-(2-carboxyprop-2-oxyimino)acetamido]-3-[(1-carbamoylmethylpyridin-ium-4-yl)thiomethyl]ceph-3-em-4-carboxylate, sodium salt when prepared by a process according to claim 5 or an obvious chemical equivalent thereof.

7. A process according to claim 1 wherein Y? represents an N-carbamoylmethylpyridinium-4-yl group and Ra and Rb together with the carbon atom to which they are attached form a cyclobutylidene group.

8. A process for preparing (6R, 7R)-7-[(z)-2-(2-aminothiazol-4-yl)-2-(1-carboxy-cyclobut-1-oxyimino)acetamido]-3-[(1-carbamoylmethylpyridinium-4-yl)thiomethyl]ceph-3-em-4-carboxylate sodium salt which comprises reacting (6R, 7R)-3-acetoxymethyl-7-[(Z)-2-(aminothiazol-4-yl)-2-(1-carboxycyclobut-1-oxyimino)-acetamido]ceph-3-em-4-carboxylic acid with l-(carbamoylmethyl)-pyridin-4-thione in the presence of sodium bicarbonate.

9. The compound (6R, 7R)-7-[(Z)-2-(2-aminothiazol-4-yl)-2-(1-carboxy-cyclobut-1-oxyimino)acetamido]-3-[(1-carbamoylmethyl-pyridinium-4-yl)thiomethyl]ceph-3-em-4-carboxylate sodium salt when prepared by a process according to claim 8 or an obvious chemical equivalent thereof.